A potential pitfall in ¹⁸O‐based N‐linked glycosylation site mapping

Abstract

A common procedure for identifying N-linked glycosylation sites involves tryptic digestion of the glycoprotein, followed by the conversion of glycosylated asparagine residues into (18)O-labeled aspartic acids by PNGase F digestion in (18)O water. The 3 Da mass tag created by this process is readily observable by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and is often used to identify the sites of N-linked glycosylation. While using this procedure, we noticed that 60% of the asparagines identified as being glycosylated were not part of the consensus sequence required for N-linked glycosylation, and thus were not biologically possible. Investigation into the source of this unacceptably high false positive rate demonstrated that even after reversed-phase cleanup and heat denaturation, the trypsin used for proteolysis was still active and led to the incorporation of (18)O into the C-termini of the peptides during the deglycosylation step. The resulting mass shift accounted for most of the false positive sites, as the database search algorithm confused it with an (18)O-labeled Asp residue near the C-terminus of a peptide. This problem can be overcome by eliminating trypsin from the solution prior to performing the deglycosylation process, by resuspending the peptides in natural abundance water following deglycosylation, or by allowing (18)O incorporation into the C-terminus as a variable modification during the database search. These methods have been demonstrated on a model protein, and are applicable to the analyses of glycoproteins that are digested with trypsin or another serine protease prior to enzymatic release of the carbohydrate side chains. This study should alert investigators in the field to this potential and unexpected pitfall and provide strategies to overcome this phenomenon.

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

“…Treated samples were then buffer-exchanged into 2.0 M urea, 50 mM Tris-HCl, pH 7.5, using Centricon spin filters as described above. Removal of residual 18 O water before enzymatic digestion is crucial, otherwise 18 O may be incorporated into the C terminus of peptides as the peptide bond is hydrolyzed during endoprotease digestion (34). Subsequent digestion with trypsin or chymotrypsin was carried out as described above.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…This method was used by Sandra et al (2007) to map glycosylation sites in Rapana venosa hemocyanine. However, Angel et al (2007), while using this procedure in a study on glycosylation of polygalacturonase-inhibiting protein isolated from Pyrus communis (pear), noticed that 60% of asparagines that were labeled were not part of a consensus sequence and thus, should not have been glycosylated. The problem was traced to residual trypsin that was used in the proteolysis still being active at the deglycosylation stage and that this enzyme was responsible for incorporation of 18 O into the C-terminus of the peptide.…”

Comprehensive characterization of the N-glycosylation status of CD44s by use of multiple mass spectrometry-based techniques

“…The +2.984 Da mass shifts were readily detectable by tandem mass spectrometry and provided a unique marker for N-linked glycosylation different from the sites of deamidation. However, PNGase F deglycosylation in H 2 18 O could also have led to incorporation of 18 O into the Ctermini of the peptides during the deglycosylation step, because of the trypsin used for proteolysis, and this incorporation could confuse the interpretation of the MS data [32]. In addition, PNGase F enzyme does not release any N-glycans when glycosites are on the first or last amino acid residue of a peptide so the protease must be carefully chosen [33].…”